Death-associated protein kinase (DAPk, DAPk-1) is a Ser/Thr kinase with a multiple domain structure (Bialik and Kimchi 2006). Its catalytic domain is located at the extreme N-terminus, and is followed by a Ca2+/calmodulin (CaM) regulatory domain. DAPk has been shown to be activated by two complementary mechanisms that serve to release the auto-inhibitory functions of this regulatory domain. One mechanism involves dephosphorylation of an inhibitory auto-phosphorylation at a critical Serine (Ser) at position 308 within the CaM regulatory domain which increases its affinity for CaM and also enhances the CaM-independent catalytic activity (Shohat, Spivak-Kroizman et al. 2001). The second involves the binding of calcium-activated CaM to the CaM regulatory domain, which results in the removal of this inhibitory domain from the catalytic cleft (Cohen, Feinstein et al. 1997) and which in turn further prevents Ser 308 negative auto-phosphorylation. Recently, the crystal structure of the binary DAPk-CaM complex has been resolved (de Diego, Kuper et al.) and the phosphatase responsible for Ser 308 dephosphorylation and DAPk activation was identified (Gozuacik, Bialik et al. 2008; Widau, Jin et al. 2010).
Other structural motifs within DAPk include a series of eight ankyrin repeats which follow the catalytic domain, after which is a region that has been shown to direct the kinase to the actin cytoskeleton (Bialik, Bresnick et al. 2004).
A putative P-loop motif which resides at amino acids 695-702 of DAPk, partially overlapping the cytoskeletal interacting domain, has been previously documented (Deiss, Feinstein et al. 1995). Yet the cellular and biochemical functions of this P-loop have not been studied so far.
The C-terminus region of DAPk contains a death domain, followed by a 17 amino acid tail rich in Ser residues (Bialik and Kimchi 2006). Several proteins have been reported to interact with the death domain of DAPk including binding to the death domain of UNC5H2 (Llambi, Lourenco et al. 2005).
An increasing amount of evidence supports the idea that DAPk works as a tumor suppressor gene in vivo. It was first noticed that DAPk mRNA and protein expression are lost in various types of cancer cell lines (Kissil, Feinstein et al. 1997). Later on, many studies have been published showing DAPk promoter hypermethylation and consequent expression silencing in tumors that had been freshly isolated from patients (Bialik and Kimchi 2004; Bialik, and Kimchi 2006). In addition, a germline mutation in DAPk was found in cases of familial chronic lymphocytic leukemia (CLL), where a single nucleotide change causes a predisposition to CLL (Raval, Tanner et al. 2007). In experimental systems it was reported that DAPk is capable of blocking tumor metastasis in vivo (Inbal, Cohen et al. 1997) and suppressing oncogenic transformation induced by c-myc and E2F in vitro (Raveh, Droguett et al. 2001). Altogether, these data established the tumor suppressive functions of DAPk, explaining why this gene is subjected to inactivation or loss during tumor development.
International Patent Application Publication No. WO 09/120,249 relates to detection of cell proliferative disorders, particularly head and neck cancer, utilizing analysis of the methylation state of targeted genes or regulatory regions of genes, including DAPk. Additional patent applications disclosing DAPk as a marker for diagnosing cancer include, inter alia, WO 09/117,346; WO 09/023,725 and WO 08/066,878.
International Patent Application Publication No. WO 08/108,964 is directed to compounds useful as selective kinase inhibitors, particularly DAPk-1 inhibitors, methods for producing such compounds and methods for treating or ameliorating chronic lymphocytic leukemia. WO 08/108,964 is further directed to methods for determining susceptibility to chronic lymphocytic leukemia in a subject includes determining a loss or reduced expression of DAPk-1 or fragments or functional equivalents thereof.
International Patent Application Publication No. WO 04/108083 relates to the treatment of malignancies by selectively inducing apoptosis. Certain aspects of the '083 publication relate to DNA methylation antagonists administered in an amount sufficient to induce expression of DAPk.
International Patent Application Publication No. WO 04/048531 provides compounds, compositions and methods for modulating the expression of DAPk-1. In particular, the '531 publication provides compounds, particularly oligonucleotide compounds, which hybridize with nucleic acid molecules encoding DAPk-1 and modulate DAPk-1 expression.
International Patent Application Publication No. WO 95/010630, to one of the inventors of the present application and others, relates to death protein (DAP) genes and DAP products for promoting death of normal or tumor cells, particularly in therapy of diseases or disorders associated with uncontrolled, pathological cell growth, e.g. cancer or psoriasis.
Gain of function of DAPk, on the other hand, has been reported to be involved in several pathologies associated with neuronal cell death such as epilepsy (Henshall, Schindler et al. 2004) and hypoxia/ischemia acute brain injury (Velentza, Wainwright et al. 2003). This was strongly supported by a recent publication showing that ischemic injury by excessive activation of NMDA glutamate receptors promotes the association of DAPk to these receptors and that disrupting this association reduces damage to the brain (Tu, Xu et al. 2010). Notably, the functional link of DAPk to different types of neuronal cell death is consistent with previous studies documenting that neurons lacking DAPk are more resistant to various death insults compared to the wild type counterparts. This was shown both in primary cultures of neurons (prepared from the DAPk knockout mice generated) and in animal model system (Pelled, Raveh et al. 2002; Schori, Yoles et al. 2002). All these studies point at the major functional role that DAPk may exert in life and death decisions of neuronal cells and make DAPk an attractive novel target for neurodegenerative drug discovery. Thus, the mechanisms of DAPk activation and regulation in cells have to be comprehended thoroughly in order for the selective inhibition of this kinase activity to be used in the treatment of such diseases.
It is noteworthy that a novel protein family was described recently, called the ROCO family (Marin, van Egmond et al. 2008). The family members, mostly comprising protein kinases, contain two common domains, the ROC (Ras of complex proteins) domain, which shares similarity with Ras and other related GTPases, and the COR (C-terminal of Roc) domain that locates downstream from the ROC domain in all members of the family. The most famous of these proteins is Leucine Repeat Rich Kinase 2 (LRRK2), a kinase implicated in Parkinson's disease. In fact, mutations in LRRK2 are the most common genetic causes of both familial and sporadic cases of late-onset Parkinson's disease (Dachsel and Farrer 2010). LRRK2's ROC domain, when bound to Guanosine-5′-triphosphate (GTP), mediates its homo-dimerization and activates the catalytic activity of the downstream kinase domain (Guo, Gandhi et al. 2007; Ito, Okai et al. 2007; Deng, Lewis et al. 2008; Greggio, Zambrano et al. 2008). Interestingly, DAPk contains a region with a significant homology to the ROC-COR domains, which includes the P-loop and cytoskeletal-targeting domain (Marin, van Egmond et al. 2008; Klein, Rovelli et al. 2009).
None of the background art, however, discloses or suggests agents which can mediate DAPk activity via the ROC domain. Such agents are, therefore, particularly useful in treating or ameliorating cell proliferative diseases (e.g., cancer) or neurodegenerative diseases such as pathologies associated with neuronal cell death.
There exists a long-felt need for more effective means of curing or ameliorating cancer and neurodegenerative diseases. The development of novel agents capable of selectively regulating DAPk activity is therefore desirable.